Several publications are referenced in this application. These references describe the state of the art to which this invention pertains, and are incorporated herein by reference.
Alzheimer's disease is a neurodegenerative disorder of the brain which is accompanied at the cellular level by a massive loss of neurons in the limbic system and in the cerebral cortex. In the brain areas affected, at the molecular level protein deposits, so-called plaques, can be detected at the molecular level, which are an essential characteristic of Alzheimer's disease. The protein occurring most frequently in these plaques is a peptide 40 to 42 amino acids in size, which is designated as Aβ-peptide. This peptide is a cleavage product of a significantly larger protein of 695 to 770 amino acids, the so-called amyloid precursor protein (APP).
APP is an integral transmembrane protein which firstly traverses the lipid bilayer. By far the largest part of the protein is extracellular, while the shorter C-terminal domain is directed into the cytosol (FIG. 1). The Aβ-peptide is shown dark-gray in FIG. 1. About two thirds of the Aβ-peptide originates from the extracellular domain and about one third from the transmembrane domain of APP.
Beside the membrane-based APP, a secreted form of the amyloid precursor protein can be detected which consists of the large ectodomain of the APP and is designated as APPsec (“secreted APP”). APPsec is formed from APP by proteolytic cleavage, which is effected by the α-secretase. The proteolytic cleavage takes place in a site of the amino acid sequence of APP which is within the amino acid sequence of the Aβ-peptide (after amino acid residue 16 of the Aβ-peptide). Proteolysis of APP by the α-secretase thus excludes the formation of the Aβ-peptide.
The Aβ-peptide can thus only be formed from APP in an alternative processing route. It is postulated that two further proteases are involved in this processing route, one protease, which is designated as β-secretase, cleaving at the N-terminus of the Aβ-peptide in the APP and the second protease, which is designated as γ-secretase, releasing the C-terminus of the Aβ-peptide (Kang, J. et al., Nature, 325, 733) (FIG. 1).
Up to now, it has not been possible to identify any of the three secretases or proteases (α-secretase, β-secretase, γ-secretase). Knowledge of the secretases, however, is of great interest, in particular in the context of investigations on Alzheimer's disease and for the identification of the proteins involved, which can then in turn be employed as targets in continuing studies. On the one hand, the inhibition of the β-secretase and in particular of the γ-secretase could lead to a reduction in the Aβ-production, on the other hand an activation of the α-secretase could increase the processing of APP in APPsec and would thus simultaneously reduce the formation of the Aβ-peptide. A transgenic C. elegans which is found in the course of such investigations is described in the unpublished German Patent Application having the reference 198 49 073.9.
There are many indications of the fact that the Aβ-peptide is a crucial factor in the occurrence of Alzheimer's disease. Inter alia, neurotoxicity of Aβ-fibrils in cell culture is postulated (Yankner, B. A. et al., (1990) Proc Natl Acad Sci USA, 87, 9020). In patients with Down's syndrome, in which APP occurs in an additional copy, the neuropathology characteristic of Alzheimer's disease also occurs even at an age of 30 years. Here, it is assumed that the overexpression of APP follows an increased conversion into the Aβ-peptide (Rumble, B. et al., (1989), N. Engl. J. Med., 320, 1446).
Perhaps the strongest indication of the central role of the Aβ-peptide are the familial forms of Alzheimer's disease. Here, mutations are found in the APP gene around the area of the β and γ-secretase cleavage sites or in two further AD-associated genes (presenilins), which in cell culture lead to a significant increase in Aβproduction (Scheuner, D. et al., (1996), Nature Medicine, 2, 864).
There are a number of indications of the fact that APP is first cleaved into the Aβ-peptide by the β-secretase during its processing in order, following this, to serve as a substrate for γ-secretase (Maruyama, K. Y. et al., (1994) Biochem. Biophys Res Commun, 202, 1517; Estus, S. et al., (1992), Science, 255, 726). The γ-secretase therefore has a crucial role in the formation of the Aβ-peptide. A demonstration of the activity of the γ-secretase which is customarily used is the detection of the Aβ-peptide, which, however, frequently turns out to be difficult.
An important reason for this is that only a small part of APP is converted into the Aβ-peptide (Simons M, et al., Neurosci (1996) 1;16(3):899–908). Moreover, the Aβ-peptide is an only very small breakage fragment of about 4 kDa and, on account of its hydrophobic character, has a great tendency to self-aggregation so that it easily precipitates under physiological conditions (Hilbich, C. et al., (1991) J. Mol. Biol., 218, 149).
The detection of the Aβ-peptide in eukaryotic cells is carried out by means of immunobiological methods such as, for example, ELISA, immunoprecipitation and Western blofting (Suzuki, N. et al., Science 1994, 27, 264(5163) 1336; Haass, C. et al., (1992) Nature, 359, 322). These processes are relatively laborious, as they involve incubation with appropriate antibodies and necessitate destruction of the cells used, which are obtained from cell culture or model organisms (inter alia C. elegans).